Biocompatible solid-phase microextraction coatings and methods for their preparation

ABSTRACT

A biocompatible coating for solid phase microextraction (SPME) of a small molecule from a biological matrix. The coating comprises SPME particles and a biocompatible polymer. The biocompatible polymer (e.g. polyacrylonitrile) reduces adsorption of proteins or macromolecules onto the SPME particles and allows the SPME particles to extract the small molecule from the matrix. A process for coating a flexible fiber with a biocompatible coating. The process comprises: coating the fiber with a suspension of SPME particles, the SPME particles being suspended in a solution of a biocompatible polymer and a solvent, the biocompatible polymer can comprise polyacrylonitrile (PAN); drying the coated fiber to remove the solvent; and curing the dried coated fiber at an elevated temperature.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/706,167, filed Feb. 15, 2007, which is a continuation ofU.S. patent application Ser. No. 11/208,933 filed Aug. 23, 2005 (nowU.S. Pat. No. 7,232,689) which is a continuation-in-part of U.S. patentapplication Ser. No. 10/506,827 filed Sep. 7, 2004 (now U.S. Pat. No.7,384,794) which is derived from International Patent ApplicationPCT/CA2003/0000311. Further this application is entitled to the benefitof and claims priority to U.S. Patent Application 60/364,214 filed Mar.11, 2002; U.S. Patent Application 60/393,309 filed Jul. 3, 2002; U.S.Patent Application 60/421,001 filed Oct. 25, 2002; U.S. PatentApplication 60/421,510 filed Oct. 28, 2002; and U.S. Patent Application60/427,833 filed Nov. 21, 2002. The entirety of each document beingincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable

FIELD OF THE INVENTION

The present invention relates to biocompatible coatings for samplingdevices used for extracting components of interest in a biologicalmatrix for further quantification or identification.

BACKGROUND OF THE INVENTION

Presently, if one wants to accurately assess the concentrations ofchemicals or drugs inside a living animal, a sample of the blood ortissue to be studied is removed from the animal and taken to ananalytical laboratory to have the chemicals of interest extracted andquantified. Typically, a first step is a pre-treatment of the sample toconvert it to a form more suitable for chemical extraction. In the caseof blood, this may be by the removal of blood cells and/or some bloodcomponents by the preparation of serum or plasma. In the case of atissue sample, this may be by many processes, including: freezing,grinding, homogenizing, enzyme treatment (e.g. protease or cellulase) orhydrolysis. Subsequently, compounds of interest are extracted andconcentrated from the processed sample. For example serum samples may besubjected to liquid-liquid extraction, solid phase extraction or proteinprecipitation, followed by drying and reconstitution in an injectionsolvent. A portion of the injection solvent is introduced to ananalytical instrument for chromatographic separation and quantificationof the components. This method produces accurate results with highspecificity for the compound of interest, but is time consuming andlabour intensive. Also, because of the large number of steps in theprocess there is a significant chance of errors in sample preparationimpacting the results. This method has good sensitivity and selectivityand accuracy for the target compounds but is limited in that thechemical balance inside the animal is disrupted during sampling. In manycases, this disruption reduces the value of the results obtained, and insome cases makes this technique inappropriate for the analysis. Wherethe removed blood volume is a high proportion of the total blood volumeof the animal, as is commonly the case when mice are used, the death ofthe animal results. This means that a different animal must be used foreach data point and each repeat. By eliminating the need for a blooddraw in this case, fewer animals would be required for testing and asignificant improvement in inter-animal variation in the results wouldbe achieved.

Alternatively, biosensors have been developed for some applications ofanalysis of chemical concentrations inside animals. In this case, adevice consisting of a specific sensing element with an associatedtransducer is implanted. The device produces a signal collected by anelectronic data logger that is proportional to the chemicals to whichthe sensor responds. The main limitations of this type of device arethat they normally respond to a spectrum of chemicals rather than havingspecificity for only one chemical. Of the spectrum of chemicals to whichthe sensor responds, some produce a greater and some a lesser response.Sensors are also susceptible to interferences where another chemicalpresent in a system interferes with the response produced by the targetchemicals. It is for these reasons that biosensors are normally limitedin terms of accuracy and precision. Additionally, biosensors aretypically not as sensitive to low chemical concentrations asstate-of-the-art, stand-alone, detectors. Such detectors, for examplemass spectrometers, are used in the above mentioned conventionalanalysis techniques and in solid phase microextraction.

Microextraction is a significant departure from conventional ‘sampling’techniques, where a portion of the system under study is removed fromits natural environment and the compounds of interest extracted andanalyzed in a laboratory environment. As with any microextraction,compounds of interest are not exhaustively removed from the investigatedsystem, and conditions can be devised where only a small proportion ofthe total amount of compound, and none of the matrix, are removed. Thisavoids disturbing the normal balance of chemical components. This couldhave a benefit in the non-destructive analysis of very small tissuesites or samples. Because extracted chemicals can be separatedchromatographically and quantified by highly sensitive analyticalinstruments, high accuracy, sensitivity and selectivity are achieved.

With current commercially available solid phase microextraction (SPME)devices, a stationary extraction phase is coated onto a fused silicafiber. The coated portion of the fiber is typically about 1 cm long andcoatings have various thicknesses. The fiber can be mounted into astainless steel support tube and housed in a syringe-like device forease of use. Extractions are performed by exposing the extraction phaseto a sample for a pre-determined time to allow sample components to comeinto equilibrium with the extraction phase. After extraction, the fiberis removed to an analytical instrument (typically a gas or liquidchromatograph) where extracted components are desorbed and analyzed. Theamount of a component extracted is proportional to its concentration inthe sample (J. Pawliszyn “Method and Device for Solid PhaseMicroextraction and Desorption”, U.S. Pat. No. 5,691,206).

To date, commercial SPME devices have been used in some applications ofdirect analysis of living systems. For example they have been appliedfor the analysis of airborne pheromones and semiochemicals used inchemical communications by insects (Moneti, G.; Dani, F. R.; Pieraccini,G. T. S. Rapid Commun. Mass Spectrom. 1997, 11, 857-862), (Frerot, B.;Malosse, C.; Cain, A. H. J. High Resolut. Chromatogr. 1997, 20, 340-342)and frogs (Smith, B. P.; Zini, C. A.; Pawliszyn, J.; Tyler, M. J.;Hayasaka, Y.; Williams, B.; Caramao, E. B. Chemistry and Ecology 2000,17, 215-225) respectively. In these cases, the living animals werenon-invasively monitored over time by assessing the chemicalconcentrations in the air around the animal, providing a convenientmeans to study complicated dynamic processes without interference.

The current commercial devices do, however, have some limitations for invivo and in vitro analysis of a biological matrix, such as blood ortissue. Firstly, the most difficult and undesirable problem is theadsorption of proteins and other macromolecules on the surface of SPMEfibers. Macromolecules are understood to be biological components with amolecular mass greater than about 10,000 atomic mass units. Thesemacromolecules constitute a diffusion barrier and decrease theextraction efficiency in subsequent experiments. In order to transferall SPME advantages to the field of in vivo and in vitro analysis ofbiological samples, it is imperative to develop new biocompatibledevices suitable for extracting compounds from biological matrices.

Devices can be made biocompatible by coating them with a biocompatiblematerial. Custom-made coatings based on polypyrrole (PPY) (Lord, H. L.;Grant, R. P.; Walles, M.; Incledon, B.; Fahie, B.; Pawliszyn, J. B.,Anal. Chem. 2003, 75 (19), 5103-5115) and poly(ethylene glycol) (PEG)(Musteata, F. M.; Musteata, M. L.; Pawliszyn, J., Clin Chem 2006, 52(4), 708-715) have been used for in vivo drug analysis. Otherbiocompatible materials include restricted access materials (RAM), ionicliquids (IL), polydimethylsiloxane (PDMS), polypyrrole, andpoly(ethylene glycol). Biocompatible membranes have also been preparedfrom polyacrylonitrile (Nie, F.-Q.; Xu, Z.-K.; Ming, Y.-Q.; Kou, R.-Q.;Liu, Z.-M.; Wang, S.-Y. Desalination 2004, 160, 43-50. Lavaud, S.;Canivet, E.; Wuillai, A.; Maheut, H.; Randoux, C.; Bonnet, J.-M.;Renaux, J.-L.; Chanard, J. Nephrology, Dialysis, Transplantation 2003,18, 2097-2104. Yang, M. C.; Lin, W. C. Journal of Polymer Research 2002,9, 201-206), polyurethane, chitosan, and cellulose.

Polymers such as polypyrroles, derivatised cellulose, polysulfones,polyacrylonitrile (PAN), polyethylene glycol and polyamides arecurrently used to prepare biocompatible membranes used for separation ofsub-micron particles in biomedical applications. PAN has been widelyused as membrane material in the fields of dialysis and ultrafiltration.It has been found that its properties can be fine-tuned by usingspecific co-monomers. The terms “polyacrylonitrile” and “PAN” are usedherein to refer to homopolymers as well as copolymers of acrylonitrilecontaining at least about 85% by weight acrylonitrile and up to about15% by weight of at least one other ethyleneically unsaturated compoundcopolymerizable with acrylonitrile. For example, PAN can be tailoredwith a reactive group for enzyme immobilization. Furthermore, someco-monomers lead to improved mechanical strength, solvent resistance,high permeation flux, and biocompatibility. Accordingly, PAN-basedmembranes have great potential for the treatment of wastewater, theproduction of ultra-pure water, hemodialysis in artificial kidneys, andbiocatalysis with separation. PAN is one of the most important polymersused in the biomedical area because of its exceptional qualities, suchas good thermal, chemical, and mechanical stability as well asbiocompatibility. Membranes made of PAN are widely used as dialyzersable to remove low to middle molecular weight proteins and for high-fluxdialysis therapy. PAN is one of the best polymers in terms ofbiocompatibility.

However, good extractive materials are generally not biocompatible andPAN is not appropriate as an extractive material for SPME.

It is, therefore, desirable to provide a biocompatible composition ableto extract small molecules from a matrix for use with solid phasemicroextraction devices, as well as a process for coating SPME fiberswith said composition.

SUMMARY OF THE INVENTION

The present invention obviates or mitigates at least one disadvantage ofprevious biocompatible compositions for solid phase microextractiondevices.

According to an aspect of the invention there is provided abiocompatible coating for solid phase microextraction (SPME) of a smallmolecule from a matrix, the coating comprising SPME particles and abiocompatible polymer, the biocompatible polymer reducing adsorption ofproteins or macromolecules onto the SPME particles and allowing the SPMEparticles to extract the small molecule from the matrix.

The SPME particles can be C-18/silica particles, RP-amide/silicaparticles, HS-F5/silica particles, normal-phase silica particles,C-1/silica particles, C-4/silica particles, C-6/silica particles,C-8/silica particles, C-30/silica particles, phenyl/silica particles,cyano/silica particles, ionic liquid/silica particles, molecularimprinted polymer particles, carboxen particles, divinylbenzeneparticles, diol/silica particles or mixtures thereof. The SPME particlescan be about 1.7 μm to about 50 μm particles, can have pore sizes fromabout 10 Å to about 200 Å, and can have a surface area of about 200 m²/gto about 800 m²/g.

The biocompatible polymer can be polyacrylonitrile (PAN), polyethyleneglycol, polypyrrole, derivatised cellulose, polysulfone or polyamide.The biocompatible polymer can be a co-polymer of polyacrylonitrile.

The matrix can be biological fluid, tissues, organs or cells. Thebiological fluid can be whole blood, plasma, serum, urine, cerebrospinalfluid, saliva or peritoneal fluid.

The small molecule can be a drug or a biomarker, the drug can be ahydrophobic or hydrophilic molecule having a molecular mass less thanabout 10,000 atomic mass units.

According to another aspect of the invention, there is provided aprocess for coating a flexible fiber with a biocompatible coating, theprocess comprising the steps of coating the fiber with a suspension ofsolid phase microextraction (SPME) particles, the SPME particles beingsuspended in a solution of a biocompatible polymer and a solvent, dryingthe coated fiber to remove the solvent, and curing the dried coatedfiber at an elevated temperature.

The SPME particles can be C-18/silica particles, RP-amide/silicaparticles, HS-F5/silica particles, normal-phase silica particles,C-1/silica particles, C-4/silica particles, C-6/silica particles,C-8/silica particles, C-30/silica particles, phenyl/silica particles,cyano/silica particles, ionic liquid/silica particles, molecularimprinted polymer particles, carboxen particles, divinylbenzeneparticles, diol/silica particles or mixtures thereof.

The biocompatible polymer can be polyacrylonitrile (PAN), polyethyleneglycol, polypyrrole, derivatised cellulose, polysulfone or polyamide.

The solvent can be dimethylformamide (DMF), dimethyl sulfoxide, NaSCN,Ca(CNS)₂, nitric acid, ethylene carbonate or mixtures thereof.

The biocompatible polymer and the solvent can be in a ratio of betweenabout 5% and 15% biocompatible polymer/solvent (w/w), the suspensioncomprising SPME particles and biocompatible polymer can be in a ratio ofabout 0.3 to about 0.7 PAN/silica (w/w).

The drying step can comprise drying the coated fiber under flowingnitrogen.

The elevated temperature can be about 180° C. to about 210° C., and thecuring step can comprise maintaining the fiber at the elevatedtemperature for about 5 seconds to about 1.5 minutes. The coating,drying and curing steps can be repeated at least once.

The process can be a continuous process or a batch process. The fibercan be a metal wire. The metal wire can be stainless steel, titanium, ora nickel-titanium alloy such as Nitinol.

According to further aspects of the invention, there is provided: afiber coated with the biocompatible coating, where the coating can beapplied as described above; a device for solid phase microextraction ofa small molecule from a matrix, the device comprising the fiber; and useof the fiber for the solid phase microextraction of a small moleculefrom a matrix.

Other aspects and features of the present invention will become apparentto those ordinarily skilled in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described, by way ofexample only, with reference to the attached Figures, wherein:

FIG. 1A shows a scanning electron micrograph image, at 100×magnification, of a fiber coated with a coating of an embodiment of thepresent invention.

FIG. 1B shows the scanning electron micrograph image at 1000×magnification.

FIG. 2 shows comparative extraction profiles over time for non-coatedSPME fibers and SPME fibers that are coated with a biocompatiblecoating.

FIG. 3 shows calibration curves for extraction of loperamide, in twodifferent matrixes, using a coating of an embodiment of the presentinvention.

DETAILED DESCRIPTION

The following detailed description illustrates the disclosure by way ofexample and not by way of limitation. The description clearly enablesone skilled in the art to make and use the disclosure, describes severalembodiments, adaptations, variations, alternatives, and uses of thedisclosure, including what is presently believed to be the best mode ofcarrying out the disclosure.

One aspect of the invention relates to coatings which can be used fordirect microextraction of small molecules from a biological matrix, suchas fluids or tissues. The biological fluids can be whole blood, serum,plasma, cerebrospinal fluid, peritoneal fluid, saliva or urine. Thetissue could be, for example, isolated cells or organs. The smallmolecules can be drugs. The small molecules can be hydrophobic orhydrophilic and should generally weigh less than 10,000 atomic massunits. The small molecules can be drugs or biomarkers. A biomarker is aphysiological substance that when present in abnormal amounts mayindicate the presence of disease. The coatings can be prepared bycovering flexible fibers with a suspension of various extractiveparticles (for example: C-18/silica, RP-amide/silica, or HS-F5/silica)in a polyacrylonitrile (PAN), polyethylene glycol, polypyrrole,derivatised cellulose, polysulfone, or polyamide solution. C-18/silicaparticles would be understood by one of skill in the art to comprisesilica particles derivatized with a hydrophobic phase, the hydrophobicbonded phase comprising octadecyl. For RP-amide-silica particles, thebonded phase comprises palmitamido-propyl. For HS-F5-silica particles,the bonded phase comprises pentafluorophenyl-propyl. The particles canbe about 1.7 to about 50 μm particles. Preferably, the particles can beabout 2 to about 20 μm particles. Preferably, the particles can be about3 to about 10 μm particles. More preferably, the particles can be about3 to about 7 μm particles. The particles can be spherical. The pore sizediameter can be about 10 to about 200 Å. Preferably, the pore size canbe about 100 to about 180 Å. The surface area can be about 200 m²/g toabout 800 m²/g. Preferably, the surface area can be about 200 m²/g toabout 300 m²/g.

It would be understood by a person of skill in the art that appropriatecoatings can be formed with other extractive particles, and particularlywith any extractive particles currently used in solid phase extractionor affinity chromatography (e.g. high pressure liquid chromatography),depending on the nature of the compound being extracted, in a similarmanner than affinity chromatography relies on different particles forseparating various compounds. For example, other particles could includesuch particles as: normal-phase silica, C1/silica, C4/silica, C6/silica,C8/silica, C30/silica, phenyl/silica, cyano/silica, diol/silica, ionicliquid/silica, molecular imprinted polymer particles, carboxen 1006 ordivinylbenzene. Mixtures of particles can also be used in the coatings.The particles can be inorganic (e.g. silica), organic (e.g. carboxen ordivinylbenzene) or inorganic/organic hybrid (e.g. silica and organicpolymer). Furthermore, a person of skill in the art would understandthat other biocompatible polymers could be used as glue or support. PANcan also be used for covering existing commercial extraction phases (forexample: carbowax/templated resin) with a biocompatible layer.

It would be readily understood by one of skill in the art that thediameter of a fiber for SPME can be of millimeter to nanometerdimensions. Preferably, the diameter of a fiber can be between 0.1millimeters and 0.6 millimeters. More preferably, the diameter of afiber can be about 0.13 millimeters (0.005 inches). The wire can beformed of any acceptable material that would be amenable for use in abiological matrix. Such material may include silica, plastic, carbon ormetal wire. Metal wires may be stainless steel, titanium, anickel-titanium alloy, or any other metal wire known to a person ofskill in the art. The flexible, inert, biocompatible nickel-titaniumalloy can be Nitinol. A metal with shape memory properties that enablethe wire to maintain straightness, even after it is coiled, can bedesirable.

Coated SPME wires can be used for in vitro analysis of drugconcentrations as well as for in vivo analysis of intravenous drugconcentrations in a living animal. Coated SPME probes for in vivoanalysis can have any combination of extractive particles coated with anappropriate biocompatible coating, such as polyacrylonitrile (PAN),polyethylene glycol, polypyrrole, derivatised cellulose, polysulfone, orpolyamide solution. Non-limiting examples of the coating include: aPAN/C-18 coating, a PAN/RP-amide coating, a polyethylene glycol/HS-F5coating, a derivatised cellulose/C-18 coating, a polypyrrole/C-30coating, a polysulfone/phenyl coating and polyamide/cyano coating.

Another aspect of the invention relates to a continuous-coating processfor producing SPME fibers coated with a biocompatible coating.Preferably, the biocompatible coating is PAN or Polyethylene glycol(PEG). In the continuous-coating process, a fiber can be wound on aspool and can be threaded through an applicator with a fixed openingthat contains a suspension of extraction particles in a biocompatiblecoating solution. The extraction particles can be C-18, RP-amide, HS-F5silica particles or any other particle listed above. Mixtures ofparticles can be used. When the particles are silica particles and thebiocompatible coating is PAN, the ratio of PAN/silica can be between 0.3and 0.7 wt/wt. The preferred ratio of PAN/silica is 0.5 wt/wt. The ratiois based on the bare weight of silica and adjusted to the phase loadingon the silica particles. The PAN/solvent solution can be between 5% and15% PAN (w/w). Preferably, the PAN/solvent solution is between about7.5% and about 12% PAN (w/w). More preferably, the PAN/solvent solutionis about 10% PAN/solvent (w/w). The solvent can be any solvent known toone of skill in the art that dissolves PAN, for example:dimethylformamide (DMF), dimethyl sulfoxide, NaSCN, Ca(CNS)₂, nitricacid, ethylene carbonate or mixtures thereof. More preferably, thesolvent can be DMF. The suspension can be coated on a length of flexiblemetal fiber. The coated fiber can be passed through a heater at anelevated temperature and connected to another reel driven by a motorthat can pull the fiber at a fixed speed. The elevated temperature canbe between about 150° C. and about 300° C. Preferably, the elevatedtemperature is between about 180° C. and about 210° C. A person of skillin the art would readily understand that PAN is fully polymerized whenit is dissolved in the solvent and as long as the solvent is fullyevaporated, the fiber is properly coated. As such, any means known to aperson of skill in the art to remove the solvent can be used to dry thecoated fibers.

In the continuous-coating process, thin multiple layers of thesuspension can be applied to the fiber until the desired coatingthickness is obtained. The advantage is that each coating layer isbonded and the coating thickness is uniform throughout the length of thefiber. When the process parameters are controlled by automation,reproducibility between fibers can be greatly improved.

Another aspect of the invention relates to a dip-coating process forproducing SPME fibers coated with a biocompatible coating. Preferably,the biocompatible coating is PAN. A dip-coating process would beunderstood by a person of skill in the art to be a batch process. Alength of fiber can be dipped into a suspension of extraction particlesin a biocompatible coating solution. The extraction particles can beC-18, RP-amide, HS-F5 silica particles or any other particle listedabove. Mixtures of particles can be used. When the particles are silicaparticles and the biocompatible coating is PAN, the ratio of PAN/silicacan be between 0.3 and 0.7 wt/wt. The preferred ratio of PAN/silica is0.5 wt/wt. The ratio is based on the bare weight of silica and adjustedto the phase loading on the silica particles. The PAN/solvent solutioncan be between about 5% and about 15% PAN (w/w). Preferably, thePAN/solvent solution can be between about 7.5% and 12% PAN (w/w). Morepreferably, the PAN/solvent solution can be about 10% PAN/solvent w/w.The solvent can be dimethylformamide (DMF), dimethyl sulfoxide, NaSCN,Ca(CNS)₂, nitric acid, ethylene carbonate or mixtures thereof. Morepreferably, the solvent can be DMF.

If desired, the coated fibers can be dried under flowing nitrogen andthen cured for about 5 s to about 1.5 min at about 180° C. to about 200°C. in order to accelerate the removal of the solvent. A person of skillin the art would readily understand that PAN is fully polymerized whenit is dissolved in the solvent and as long as the solvent is fullyevaporated, the fiber is properly coated. As such, any means known to aperson of skill in the art to remove the solvent can be used to dry thecoated fibers.

The wires can be pre-processed before the coating process in order toclean and roughen the surface. Pre-processing can be accomplished bywashing with acetone, etching for 1 min in concentrated hydrochloricacid, washing the wire with water and/or thoroughly cleaning the wire bysonication in water. Prior to use, the coated fibers can be conditionedin a water:methanol 50:50 wash for 30 min. Conditioning the C-18 basedcoatings with water or higher proportion of methanol can lead to worsereproducibility. Other coatings, however, can require only a very briefconditioning step (less than 5 min), or even none at all.

EXAMPLE 1 Dip Coating

Particles commonly used as HPLC stationary phases (0.47 g of C-18,RP-amide, or HS-F5 particles) were brought into suspension with 2 g of asolution made up of 10% w/w PAN in DMF. SPME coatings with a length of1.5 cm were prepared by applying a uniform layer of slurry of PAN anddifferent particles on the surface of stainless steel wires, allowing todry under flowing nitrogen, and finally curing for about 1.5 min at 180°C. The SPME coating was applied by dipping the wires into the slurry andremoving them slowly.

EXAMPLE 2 PAN as a Membrane

Existing fibers with conventional extraction phases(CW/TPR—carbowax/templated resin, from Supelco, PA) were coated with PANby dipping them for 2 min in a solution of 10% PAN in DMF. Subsequently,the fibers were removed slowly from the solution, allowed to dry underflowing nitrogen, and finally cured by a short exposure (5 s) to a flowof nitrogen at 200° C.

EXAMPLE 3 Continuous Coating

Wire was coiled on a first reel and threaded through an applicatorfilled with a coating suspension. The wire was then threaded through aheater and attached to a take-up reel. The wire was drawn through theboth the applicator and heater at a set speed. The thickness of thecoating was measured and additional coatings were applied by switchingthe positions of the first reel and take-up reel, and repeating theprevious coating, drying and switching steps until a desired thicknessis achieved.

EXAMPLE 4 Analysis by Scanning Electron Microscopy

For SEM imaging, the fibers were cut into 7 mm long pieces, coated withgold (˜10 nm) and analyzed using a LEO 1530 Emission Scanning ElectronMicroscope at the Waterloo Watlab Facility. The SEM images of PAN/C-18coatings (FIG. 1) demonstrate that the particles are completely coveredwith PAN and are homogeneously distributed within the coating.

SEM was also used to estimate the average thickness of each coating,which was found to be 60-62 μm. No swelling of the coating was observedduring analysis time (extraction up to about 2 h and desorption forabout 15 min).

EXAMPLE 5 Analysis by X-Ray Photoelectron Spectroscopy

XPS (X-ray photoelectron spectroscopy) analyses were performed by usinga multi-technique ultra-high vacuum Imaging XPS Microprobe system(Thermo VG Scientific ESCALab 250) equipped with a hemisphericalanalyzer with a mean radius of 150 mm and a monochromatic Al—K□ (1486.60eV) X-ray source. The spot size for the XPS analysis used for thepresent work was approximately 0.5 mm by 1.0 mm. The samples weremounted on a stainless steel sample holder with double-sided carbontapes. The sample was stored in vacuum (2×10⁻⁸ mbar) in the load-lockchamber of the Imaging XPS Microprobe system overnight to remove anyremaining moisture before introduction into the analysis chambermaintained at 2×10⁻¹⁰ mbar. A combination of low energy electrons andions was used for charge compensation on the non-conducting coatingmaterial during the analysis conducted at room temperature. Averages offive high resolution XPS scans were performed for each element ofinterest (C, N, O, S). Curve fitting was performed using CasaXPS VAMASProcessing Software and the binding energies of individual elements wereidentified with reference to the NIST X-Ray Photoelectron SpectroscopyDatabase.

All investigated fibers were exposed to undiluted human plasma at 37° C.for 1 h (this is considered a rigorous biocompatibility test). They werethen briefly washed with phosphate buffer and deionized water and driedin nitrogen before analysis. Survey scans and high resolution XPS scanswere used to determine the atomic percentages of the surfaces before andafter exposure to plasma, as described in Example 8.

EXAMPLE 6 Analysis by LC/MS

Stock solutions of drugs (diazepam, verapamil, warfarin, nordiazepam,loperamide, and lorazepam as internal standard) with a concentration of1 mM were prepared in a water:methanol 1:1 mixture and kept refrigeratedat 4° C. (in 2 mL silanized vials).

Human plasma (in 2 mL polypropylene vials with EDTA as anti-clottingagent) was stored at −20° C. until analysis. For analysis, plasma wasthawed at room temperature and aliquots of 1.5 mL plasma weretransferred into clean vials. Appropriate amounts of stock drug solutionwere added to obtain final concentrations of drug in the range 1 nM-50μM, followed by vortex mixing for 1 minute. Samples and standards in PBS(phosphate buffer saline) were similarly prepared, to a finalconcentration in the range 0.1 nM-5 μM.

The time required for the drugs to reach equilibrium between the sampleand the SPME fiber, for plasma and PBS samples at 2400 rpm vortexstirring and room temperature, was determined for all target compounds(diazepam, verapamil, and nordiazepam 5E-7M; warfarin 5E-6M; loperamide5E-8M) by measuring the amount of compound extracted at different timepoints. Although the concentration of the sample analyzed by SPME has noimpact on the extraction time profile and equilibration time, theagitation conditions, coating thickness (especially for liquidcoatings), distribution constant, and diffusion coefficient of theanalyte play very important roles in determining an experimentalequilibration time. While the theoretical equilibration time isinfinite, the experimental equilibration time can be considered to bethe time required to extract at least 95% of the theoretical maximum.

To minimize the errors caused by different sampling times, theextraction time should be equal to or longer than the experimentalequilibrium time. The experimentally determined equilibration time wasfound to be between 4 and 55 min in most cases. No significantdifference was observed when the equilibration profile in PBS wascompared to the equilibration profile in plasma. When the target drugswere analyzed in mixtures, an extraction time corresponding to themaximum equilibration time was used.

When existing commercial coatings were covered with a layer of PAN, theequilibration time remained essentially unchanged. The mechanicalstability of the fibers coated with PAN can be significantly improved:while original fibers can be used for 20 extractions before they breakdown, those coated with PAN can last for more than 50 extractions. Inaddition to improved biocompatibility and durability, the PAN coatedfibers offer almost the same extraction capacity as the non-coatedfibers (FIG. 2).

For extraction, samples were placed on a digital vortex platform and theextracting phase of the SPME fiber was immersed in the sample for aprecise period of time, as determined above. Subsequently, the fiber wasthen briefly rinsed with water, and desorbed for analysis. The lowestcarryover and the sharpest chromatographic peaks for the investigateddrugs were obtained for a desorption time of 15 min, vortex stirring at2400 rpm, and with a desorption solution prepared fromacetonitrile:water:acetic acid (50:49:1). Unless otherwise specified,the sample volume was 1.5 mL and the fiber was desorbed for 15 minutesin an insert with 60 μL desorption solution containing lorazepam asinternal standard (50 ng/mL).

Successful coupling of SPME with HPLC is dependent on the efficiency ofthe desorption step. Desorption can be effected on-line (manualintroduction of the fiber into a desorption chamber) or off-line (in avial or 96-well plate).

The carryover was found to be well below 3% (with three exceptions outof twenty determinations). For highly sensitive analyses, desorption isusually followed by solvent evaporation and reconstitution in a lowervolume of solvent suitable for direct HPLC analysis. Nevertheless,desorption in 60 μL solvent was found to be entirely suitable. Ifrequired, the carryover can be further decreased by using larger volumesof desorption solution or longer desorption time.

All reproducibility, reusability, extraction efficiency, and calibrationexperiments were performed at equilibrium in similar conditions,following the general procedure for new SPME methods. Calibration curveswere constructed by spiking PBS or human plasma with drug concentrationsin the range of 0.5 nM-50 μM, which generally covers the therapeuticconcentrations. All extractions and desorptions were performed manually.

LC-MS (liquid chromatography coupled with mass spectrometry) analyseswere performed using an Agilent 1100 series liquid chromatograph(Agilent Technologies, Palo Alto, Calif.), equipped with a vacuumsolvent degassing unit, a binary high pressure gradient pump, anautosampler, a column thermostat and a variable wavelength UV-VISdetector coupled on-line with an Agilent 1100 series MSD singlequadrupole instrument with atmospheric pressure electrospray-ionization(ESI). High purity nitrogen used as nebulizing and drying gas wasobtained from an in-house generator.

Chromatographic separations were carried out on a Discovery® C18 column(5 cm×2.1 mm, 5 μm particles, from Supelco), guarded by an on-linefilter (0.2 μm). Data were collected and analyzed using the CHEMSTATIONsoftware from Agilent Technologies.

LC and ESI-MS conditions were as follows: column temperature 25° C.,mobile phase acetonitrile: 20 mM ammonium acetate pH=7.0 with gradientprogramming (initial composition—10:90, ramped to 80:20 over 6 min andmaintained until the end of the run), flow rate 0.25 mL min⁻¹, nebulizergas N₂ (35 psi), drying gas N₂ (13 L min⁻¹, 300° C.), capillary voltage3500 V, fragmentor voltage 80 V, quadrupole temperature 100° C.,positive ionization mode. Total run time was 9 min.

For optimization experiments, scan mode in the range 100-1500 amu wasused; for quantification experiments, selected ion monitoring is used,with a scan time of 0.42 s/cycle and a dwell time of 65 ms. Thefollowing positive ions were monitored: diazepam, m/z 285.1; verapamil,m/z 455.3; warfarin, m/z 309.1; nordiazepam, m/z 271.1; loperamide, m/z477.3; lorazepam, m/z 321.0. All other parameters of the mass-selectivedetector were automatically optimized using a calibration standard.Lorazepam was used as an internal standard for compensation ofvariations in the injection volume (20 μL).

EXAMPLE 7 Sterilization

Sterilization may be desired if the microextraction devices are to beused for in vivo experiments. Current sterilization methods includeheat, steam, chemical (ethylene oxide, alcohols, aldehydes), andradiation.

The new coatings were tested for extraction efficiency before and afterchemical and steam sterilization. For chemical sterilization, the fiberswere immersed in alcohol (methanol or ethanol) for 30 minutes and thenallowed to dry. Sterilization by steam was performed in an autoclave at121° C. and 15 psi for 30 minutes.

No change in extraction efficiency was observed upon sterilization withalcohols, as this step is similar to the conditioning step (beforeextraction). In the case of sterilization in an autoclave, the proposedcoatings showed no sign of deterioration (as determined from opticalmicroscope images). This was expected since PAN coatings are known towithstand GC-injector temperatures (>250° C.). Although no signs ofbreakdown were observed, the extraction capacity decreased byapproximately 15% after sterilization, possibly because of the combinedeffect of heat and water vapors on the fused silica particles.

EXAMPLE 8 Biocompatability

Many methods have been applied for the study of biocompatibility,ranging from the simple visual inspection to the most sensitive atomicforce microscopes. Nevertheless, only a few methods are widely used andrecognized: XPS, atomic force microscopy, surface plasmon resonance, andcompetitive ELISA (enzyme linked immunosorbent assay).

XPS or electron spectroscopy for chemical analysis (ESCA) is one of themost common types of spectroscopic methods for analysis of surfaces. Thesampling depth for this method is approximately 1-30 nm (up to 100 nmmean-free pass), which encompasses a surface region highly relevant forbio-interactions.

The biocompatibility of various coatings was tested by XPS. A materialis considered biocompatible if the amount of nitrogen and sulfur on thesurface does not increase significantly after contact with a biologicalsystem. After exposure of PAN-based coatings to plasma, the amount ofnitrogen and carbon on the surface generally decreases, accompanied byan increase in the amount of oxygen (Table 1). These observationssuggest that most of the molecules adsorbed from human plasma contain ahigh percent of oxygen (usually because of non-specific adsorption),while their nitrogen content is lower than that of plasma proteins. Evenmore conclusive from a biocompatibility point of view is the amount ofsulfur on the surface, since sulfur is naturally present in proteins butabsent from the investigated SPME coatings. When compared to RAM andPPY, materials regarded as highly biocompatible, the new coatings basedon PAN showed a much lower increase in sulfur.

TABLE 1 Atomic composition obtained by XPS for selected proteins andcoatings (before and after exposure to human plasma); hydrogen is notreported, as it is not detectable by XPS. C % N % O % S % (RSD < (RSD <(RSD < (RSD < Protein/Coating 5%) 5%) 10%) 15%) Human serum albumin 63.316.9 19.0 0.9 Fibrinogen 62.8 18.0 18.8 0.5 PAN coating (bp*) 78.2 17.64.0 0.0 PAN coating (ap**) 73.5 15.1 11.3 0.0 PAN/C-18 (bp*) 77.0 17.25.7 0.0 PAN/C-18 (ap**) 73.6 13.9 12.5 0.0^(§) PAN/RP-amide (bp*) 78.317.0 4.5 0.0 PAN/RP-amide (ap**) 68.6 15.5 15.8 0.0 PAN/HS-F5 (bp*) 79.320.1 0.4 0.0 PAN/HS-F5 (ap**) 70.9 15.2 13.7 0.0 PAN/RAM (bp*) 78.9 20.40.5 0.0 PAN/RAM (ap**) 72.6 16.3 10.4 0.5 PPY (bp*) 61.0 3.8 35.2 0.0PPY (ap**) 69.7 12.4 17.6 0.3 *bp = before exposure to human plasma **ap= after exposure to human plasma ^(§)the experimental value was 0.04,below the limit of quantitation of 0.1%

The biocompatibility test based on XPS suggests that the mostbiocompatible PAN-based coatings are PAN/RP-amide and PAN/HS-F5,followed closely by PAN/C-18. Furthermore, the newly developed PAN-basedcoatings were inspected under the microscope after five minutes exposureto human plasma and whole mouse blood (without anti-clotting agents),and no clot adhesion to the coating was observed.

EXAMPLE 9 Drug-Plasma Protein Binding

Various SPME coatings were investigated by studying the extraction andseparation of drugs from human plasma. As shown in FIG. 3, a very goodlinear relationship was obtained for a seven point calibration (n=3).FIG. 3 also indicates that drug binding to plasma proteins changes theamount of drug available for extraction and results in differentcalibration slopes for plasma and PBS.

The linear range covered more than three orders of magnitude for mostdrugs, with the exception of warfarin, where the linear range spannedover two orders of magnitude. The full details are shown in Table 2.

TABLE 2 Linear ranges for SPME-based analytical method. Linear RangePAN/C-18 PAN/RP-amide (moles/L) PBS Plasma PBS Plasma Diazepam 1E−9 −>2E−6 1E−8 −> 1E−5 3E−9 −> 1E−6 5E−8 −> 1E−5 Verapamil 1E−9 −> 1E−6 5E−9−> 5E−6 2E−9 −> 4E−7 2E−8 −> 4E−6 Warfarin 2E−8 −> 5E−6 2E−7 −> 5E−52E−8 −> 4E−6 1E−6 −> 4E−5 Nordiazepam 1E−8 −> 5E−6 1E−7 −> 2E−5 7E−9 −>2E−6 2E−7 −> 2E−5 Loperamide 1E−9 −> 2E−7 5E−9 −> 2E−6 2E−9 −> 2E−7 2E−8−> 2E−6

The determination of plasma protein binding by SPME is based ondetermining the free concentration of drug in the presence of plasmaproteins. Briefly, the percentage of drug binding to plasma proteins(PPB) is calculated from the total and free concentration of drug:

$\begin{matrix}{{P\; P\; B\mspace{14mu} \%} = {{\frac{C_{{total}\mspace{14mu} {plasma}} - C_{{free}\mspace{14mu} {plasma}}}{C_{{total}\mspace{14mu} {plasma}}} \cdot 100} = {( {1 - \frac{C_{{free}\mspace{14mu} {plasma}}}{C_{{total}\mspace{14mu} {plasma}}}} ) \cdot 100}}} & (1)\end{matrix}$

where C_(total plasma) is the total concentration of drug in plasma andC_(free plasma) is the free concentration of drug in plasma.

Considering that the total drug concentration is directly proportionalto the slope of the drug calibration curve in PBS and the freeconcentration is directly proportional to the slope of plasmacalibration, Equation 1 becomes:

$\begin{matrix}{{P\; P\; B\mspace{14mu} \%} = {100 \cdot ( {1 - \frac{{slope}\mspace{14mu} {calibration}\mspace{14mu} {plasma}}{{slope}\mspace{14mu} {calibration}\mspace{14mu} P\; B\; S}} )}} & (2)\end{matrix}$

Equation 2 was applied for the determination of drug plasma proteinbinding for the five test drugs, and the results are presented in Table3. Only the most reproducible coatings were used, and the resultscorrelate very well with previously published values.

TABLE 3 Experimental and literature drug plasma protein binding values.Plasma Protein PAN/C-18 PAN/RP-amide Literature Binding % 0.01″, 60 μm0.01″, 60 μm Values (range) Diazepam 98 99 96-98 Verapamil 96 96 88-98Warfarin 99 99  98-100 Nordiazepam 98 98 97-98 Loperamide 96 97 95-97

For the extraction efficiency test, the PAN coatings based on C-18,RP-amide and HS-F5 showed, on average, much higher extraction efficiencytowards the investigated drugs: ˜90 times more than PPY, ˜50 times morethan RAM or PDMS coatings, and ˜20 times more than commerciallyavailable CW/TPR.

The above-described embodiments of the present invention are intended tobe examples only. Alterations, modifications and variations may beeffected to the particular embodiments by those of skill in the artwithout departing from the scope of the invention, which is definedsolely by the claims appended hereto.

1. A biocompatible coating for solid phase microextraction (SPME) of asmall molecule from a matrix, the coating comprising: SPME particles;and a biocompatible polymer; wherein the biocompatible polymer reducesadsorption of proteins or macromolecules onto the SPME particles andallows the SPME particles to extract the small molecule from the matrix.2. The biocompatible coating according to claim 1, wherein the SPMEparticles are selected from the group consisting of C-18/silicaparticles, RP-amide/silica particles, HS-F5/silica particles,normal-phase silica particles, C-1/silica particles, C-4/silicaparticles, C-6/silica particles, C-8/silica particles, C-30/silicaparticles, phenyl/silica particles, cyano/silica particles, ionicliquid/silica particles, molecular imprinted polymer particles, carboxenparticles, divinylbenzene particles, diol/silica particles and mixturesthereof.
 3. The biocompatible coating according to claim 2, wherein theSPME particles are selected from the group consisting of C-18/silicaparticles, RP-amide/silica particles and HS-F5/silica particles.
 4. Thebiocompatible coating according to claim 1, wherein the biocompatiblepolymer is selected from the group consisting of polyacrylonitrile(PAN), polyethylene glycol, polypyrrole, derivatised cellulose,polysulfone and polyamide.
 5. The biocompatible coating according toclaim 4, wherein the biocompatible polymer is polyacrylonitrile (PAN).6. The biocompatible coating according to claim 1, wherein the SPMEparticles are about 1.7 μm to about 50 μm particles.
 7. Thebiocompatible coating according to claim 1, wherein the SPME particleshave a pore size from about 10 Å to about 200 Å.
 8. The biocompatiblecoating according to claim 7, wherein the SPME particles have a poresize from about 80 Å to about 180 Å.
 9. The biocompatible coatingaccording to claim 1, wherein the SPME particles have a surface area ofabout 200 m²/g to about 800 m²/g.
 10. The biocompatible coatingaccording to claim 1, wherein the polymer is a co-polymer ofpolyacrylonitrile.
 11. The biocompatible coating according to claim 1,wherein the matrix is selected from the group consisting of biologicalfluid, tissues, organs and cells.
 12. The biocompatible coatingaccording to claim 11, wherein the biological fluid is whole blood,plasma, serum, urine, cerebrospinal fluid, saliva or peritoneal fluid.13. The biocompatible coating according to claim 1, wherein the smallmolecule is a drug or a biomarker.
 14. The biocompatible coatingaccording to claim 12, wherein the drug is a hydrophobic or hydrophilicmolecule having a molecular mass less than about 10,000 atomic massunits.
 15. A process for coating a flexible fiber with a biocompatiblecoating, the process comprising the steps of: coating the fiber with asuspension of solid phase microextraction (SPME) particles, the SPMEparticles being suspended in a solution of a biocompatible polymer and asolvent; drying the coated fiber to remove the solvent; and curing thedried coated fiber at an elevated temperature.
 16. The biocompatiblecoating according to claim 15, wherein the SPME particles are selectedfrom the group consisting of C-18/silica particles, RP-amide/silicaparticles, HS-F5/silica particles, normal-phase silica particles,C-1/silica particles, C-4/silica particles, C-6/silica particles,C-8/silica particles, C-30/silica particles, phenyl/silica particles,cyano/silica particles, ionic liquid/silica particles, molecularimprinted polymer particles, carboxen particles, divinylbenzeneparticles, diol/silica particles and mixtures thereof.
 17. Thebiocompatible coating according to claim 16, wherein the SPME particlesare selected from the group consisting of C-18/silica particles,RP-amide/silica particles and HS-F5/silica particles.
 18. Thebiocompatible coating according to claim 15, wherein the biocompatiblepolymer is selected from the group consisting of polyacrylonitrile(PAN), polyethylene glycol, polypyrrole, derivatised cellulose,polysulfone and polyamide.
 19. The biocompatible coating according toclaim 18, wherein the biocompatible polymer is polyacrylonitrile (PAN).20. The process according to claim 15, wherein the solvent is selectedfrom the group consisting of: dimethylformamide (DMF), dimethylsulfoxide, NaSCN, Ca(CNS)₂, nitric acid, ethylene carbonate and mixturesthereof.
 21. The process according to claim 20, wherein the solvent isdimethylformamide (DMF).
 22. The process according to claim 15, whereinthe solution comprises the biocompatible polymer and the solvent beingin a ratio of between about 5% and 15% biocompatible polymer/solvent(w/w).
 23. The process according to claim 22, wherein the biocompatiblepolymer/solvent ratio is between about 7.5% and 12% (w/w).
 24. Theprocess according to claim 23, wherein the biocompatible polymer/solventratio is about 10% (w/w).
 25. The process according to claim 15, whereinthe suspension comprises SPME particles and biocompatible polymer beingin a ratio of about 0.3 and about 0.7 PAN/silica (w/w).
 26. The processaccording to claim 25, wherein the PAN/silica ratio is about 0.5 (w/w).27. The process according to claim 15, wherein the drying step comprisesdrying the coated fiber under flowing nitrogen.
 28. The processaccording to claim 15, wherein the elevated temperature is about 180° C.to about 210° C., and the curing step comprises maintaining the fiber atthe elevated temperature for about 5 seconds to about 1.5 minutes. 29.The process according to claim 15, wherein the coating, drying andcuring steps are repeated at least once.
 30. The process according toclaim 15, wherein the process is a continuous process.
 31. The processaccording to claim 15, wherein the process is a batch process.
 32. Theprocess according to claim 15, wherein the fiber is a metal wire. 33.The process according to claim 32, wherein the metal wire is stainlesssteel, titanium, a nickel-titanium alloy.
 34. The process according toclaim 33, wherein the nickel-titanium alloy is Nitinol.
 35. A fibercoated with the biocompatible coating as defined in claim
 1. 36. Adevice for solid phase microextraction of a small molecule from amatrix, the device comprising the fiber as defined in claim
 35. 37. Useof the fiber as defined in claim 35 for the solid phase microextractionof a small molecule from a matrix.
 38. A fiber coated according to theprocess of claim
 15. 39. A device for solid phase microextraction of asmall molecule from a matrix, the device comprising the fiber accordingto claim
 38. 40. Use of the fiber as defined in claim 38 for the solidphase microextraction of a small molecule from a matrix.